A robust system of hybridogenesis that increases genetic variability and promotes evolutionary succession in greenlings (Teleostei: Hexagrammidae, genus Hexagrammos): Regeneration of a new hemiclonal lineage

Unisexual hybrids that reproduce either clonally or hemiclonally are considered to be evolutionarily short-lived as they lack the ability to reduce deleterious mutations and increase genetic diversity. In the greenling (Teleostei: Hexagrammidae, genus Hexagrammos), unisexual hybrids that produce haploid eggs containing only the H. octogrammus (maternal species) genome generate hemiclonal offspring by fertilization with haploid sperm of H. agrammus (paternal species). When hemiclonal hybrids are backcrossed to a male of the maternal species, the offspring (BC-Hoc) are phenotypically similar to the maternal species and produce recombinant gametes through conventional meiosis. BC-Hoc (recombinant generation) individuals referred to as carriers harbor the genetic factor for hybridogenesis, thereby facilitating the production of new hemiclonal lineages through hybridization. Previous studies based on field research have suggested that the carriers produced by two-way backcrossing (mating pattern in which hemiclonal hybrids are backcrossed with both parental species) may overcome the evolutionary dead end imposed by the lack of recombination. The present study verified this hypothesis by regenerating a newly hemiclonal lineage through artificial hybridization. To clarify the genetic mode of hybrids produced by crosses between BC-Hoc and Hag, mature eggs were obtained from 16 individuals and fertilized with either Hag or Hoc sperm. Hybridogenesis was confirmed in one of the 16 individuals. Based on the low occurrence rate, these findings suggest that hemiclonal lineages can be regenerated, and that the hemiclonal factors are likely distributed across multiple genes on different chromosomes. The findings provide important evidence for the retention of a robust system for increasing genetic variability and maintaining evolutionary succession in unisexual hybrids that reproduce hemiclonally.


Introduction
Interspecific hybridization is observed frequently in fish [1,2].While interspecific hybrids are often infertile due to pairing incompatibilities between homologous chromosomes, some hybrid lineages maintain their populations by switching between bisexual and unisexual modes of reproduction by employing mechanisms such as clonal reproduction (parthenogenesis and gynogenesis) or hemiclonal reproduction (hybridogenesis) [3][4][5][6][7].In clonal reproduction, the female produces unreduced diploid or triploid eggs, and sperm are used only to trigger embryogenesis [5].Therefore, the male makes no biological or genetic contribution to the offspring.Conversely, in hemiclonal reproduction (hybridogenesis), the paternal genomes also play a role in somatic growth during embryogenesis after fertilization, which means that the offspring retain both the maternal and paternal genomes in their somatic cells.However, during oogenesis within the gonadal tissue, one parental genome, typically the paternal genome, is excluded from germ cells, which culminates in the production of haploid eggs that carry only the other parental genome [8][9][10].
Without recombination, unisexual hybrids are expected to become extinct in 10,000 to 100,000 generations because they accumulate deleterious mutations in their genomes and genetic diversity is limited [11][12][13].There are several considerations regarding the mechanisms by which unisexual organisms produce genetic variation, such as the partial incorporation of paternal chromosomes and increasing ploidy [3,14].However, neither the mechanisms that reduce the extinction risk and break the evolutionary dead end in unisexual vertebrates, nor the conditions for creating new unisexual reproductive lineages are currently well understood [15].
The "balance hypothesis" [16,17] is a well-known explanation for why unisexual reproduction occurs.According to this hypothesis, unisexual lineages occur in very limited cases, such as when the genetic dissimilarity between two sexual species is extensive enough to disrupt typical gametogenesis, but the hybrids remain viable and sufficiently similar to contribute towards the development of a stable phenotype [16,17].However, recent studies have suggested that unisexual reproduction might be caused not only by the extent of the genomic dissimilarity between species, but also by "genetic factors" to induce hybridogenesis [15,[18][19][20].
The genus Hexagrammos includes six species of greenlings that are widespread in the coastal waters of the North Pacific Ocean [21].One boreal species, the masked greenling H. octogrammus (hereafter Hoc), and two temperate species, the fat greenling H. otakii (Hot) and the spotty belly greenling H. agrammus (Hag), have sympatric distributions in coastal areas of southern Hokkaido (Japan) and Primorsky Krai (Russia) [22].The hybrid zone that is inhabited by these three Hexagrammos species contains two unisexual hybrids that have been reported to reproduce hemiclonally [19].These hemiclonal hybrids produce haploid eggs containing only the H. octogrammus genome (maternal ancestor) and generate diploid hemiclonal offspring by fertilization with haploid sperm from either H. agrammus or H. otakii (paternal species) [19].Mating experiments in Hexagrammos hybrids have shown the existence of certain genetic factors that induce hybridogenesis [19].Therefore, the hemiclonal Hoc* genome of the unisexual hybrids is differentiated from the Hoc genome of the pure species, and the two hemiclonal hybrids are denoted as Hoc*/Hag and Hoc*/Hot ("*" indicates that the genetic factors required for inducing hybridogenesis are present).Hoc*/Hoc (hereafter BC-Hoc*) individuals, which are produced by maternal backcrossing of Hoc*/Hag hybrids with Hoc individuals, have been shown to produce recombinant gametes despite having inherited a Hoc* genome from a hemiclonal hybrid (Fig 1) [23].This suggests that the hemiclonal genes are not expressed between homologous genomes, but only when heterologous genomes are mated.
In our previous study, we elucidated a mechanism for reducing extinction risk by two-way backcrossing in the field [20].The "recombination generation" produced by two-way backcrossing has been suggested to be important for overcoming the evolutionary dead-end of the hemiclonal lineage.Recombinant generations which carried hemiclonal genes in the gene pool of the maternal species would behave as Hoc in wild and seemed to be morphologically undistinguishable from the maternal species.Here, we examined whether BC-Hoc* and the new hybrid lineages produced from BC-Hoc* were not morphologically different to the Hoc and hybrids in wild populations.Finally, we try to produce a new hemiclonal lineage from BC-Hoc* that we generated by artificial fertilization to experimentally confirm that multiple genetic factors are implicated in expression of hybridogenesis and that evolutionary dead-ends can be overcome by shuffling the maternal genome (Fig 1).

Fish sampling and artificial fertilization
The two Hexagrammos species (Hoc and Hag) and the natural hybrid (Hoc*/Hag) were caught by traps and/or fishing rods in the vicinity of the Usujiri Fisheries Station (N41˚57', E140˚58') of the Field Science Center for Northern Biosphere, Hokkaido University in southern Hokkaido, Japan, from 2010 to 2016.Until artificial fertilization, the captured fishes were kept in 500 L tanks containing concrete blocks which served as hiding places.The tanks were circulated with fresh seawater that was the same temperature as that of the natural environment.The fishes were fed daily with a diet consisting of Japanese anchovy, krill and artificial pellets (Otohime, Nishinmarubeni Co., Japan).In 2010, to produce BC-Hoc*, mature eggs from Hoc*/Hag hybrids were artificially fertilized with sperm from Hoc individuals.After two years (2012), when the BC-Hoc* individuals were mature, mature eggs of BC-Hoc* females were fertilized artificially with sperm from Hag individuals to produce BC-Hoc* × Hag individuals (A × B indicates A: female × B: male artificial crossing).By 2016, a total of 21 mature BC-Hoc* × Hag individuals (16 females and 5 males) had been raised.To confirm whether BC-Hoc* × Hag produced hemiclonal or recombined eggs, eggs from 16 BC-Hoc* × Hag females were fertilized with sperm from Hag or Hoc males.A total of seven larvae from each of the 16 clutches were then subjected to microsatellite DNA analysis.
Artificial fertilization was performed as described previously [22,23] and the clutches were incubated for 20-23 days at 11-15˚C until hatching.Tissue samples of hatched larvae and parental fishes for genetic analysis were preserved in 99% ethanol at -20˚C.Samples of hatched larvae for genetic analysis were anesthetized using ethyl m-aminobenzoate methanesulfonate (Nacalai Tesque Inc., Kyoto, Japan) and fixed.Parental fishes used for artificial fertilization were housed for use in further research.Naturally dead BC-Hoc* and BC-Hoc* × Hag hybrids were fixed in 10% seawater formalin and preserved in 50% 2-propanol for morphological analysis.

Ethics statement
The experimental procedures involving fish were approved by the Institutional Animal Care and Use Committee (The Regulations of Animal Experimentation at Hokkaido University), Permit number 26-1, according with directives from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Morphological analysis
To ascertain the morphological identity of the BC-Hoc* to Hoc and BC-Hoc* × Hag to Hoc*/ Hag hybrids, we counted seven parameters and measured nine parameters for morphological characters (listed in Table 1) for BC-Hoc* and BC-Hoc* × Hag individuals preserved in 2-propanol.Transversal line scales were counted only in BC-Hoc*.Morphometric data from 10 adult BC-Hoc* and 8 BC-Hoc* × Hag individuals were compared to those from 37 Hoc, 13 Hag and 4 Hoc*/Hag individuals by referring to [19].To visualize the differences among the five lineages, the principal component analysis (PCA) was performed using five counts, excluding Transversal line scales and Lateral lines, and nine measurements converted to percentages of the standard length.Counts, measurements, and descriptive terminology followed [19,24].Since the tips of the fins of the fish bred in captivity showed signs of wear due to abrasion with the tank, and since no interspecific (lineage) differences in the length of each fin were observed in previous studies [19], fin length was not used as a morphometric character in this study.The principal Component Analysis was performed using R version 4.2.2 (R Core Team 2020).

Detection of hemiclonal hybrids by microsatellite DNA
Seven microsatellite loci identified as informative in previous studies [19,20,25] were used to clarify the inheritance pattern of the gametes that BC-Hoc* × Hag hybrids produced (S1 Table ).First, to clarify the genesis mode of each clutch, one primer sets for microsatellite loci were selected by focusing on loci where the parental fish exhibited heterozygosity.If two maternal alleles appeared alternatively in a clutch, then the BC-Hoc* × Hag hybrids was considered to have produced a recombinant gamete, and the analysis of such clutches was terminated.In all primer sets, if all offspring shared only a single maternal allele, then the BC-Hoc* × Hag hybrids was considered to have hybridogenesis.
Total genomic DNA was extracted using a Quick Gene DNA tissue kit S (Fujifilm, Japan) according to the manufacturer's instructions and stored in a refrigerator at 4˚C until use.The PCR mixes contained 12.5 μl of EmeraldAmp PCR Master Mix (Takara Bio Inc., Japan), 0.25 μl of each primer (20 μM), 1 μl of template DNA (50-100 ng/μl), and 11 μl of water to give a final volume of 25 μl.The PCR profiles for the seven regions were the same as those reported previously [20,25].Genotyping was performed by electrophoresis on 8.25% polyacrylamide gels stained with SYBR Green II (Takara Bio Inc., Japan).

Morphological analysis
In the principal component analysis (PCA), the first, second and third PCs accounted for 37.

Detection of hemiclonal hybrids
To detect heterozygosity, 109 offspring of the 16 BC-Hoc* × Hag hybrids were genotyped using microsatellite loci.The resulting inheritance patterns exhibited by the progeny of the 16 BC-Hoc* × Hag hybrids are shown in Tables 2 and 3.In one out of 16 clutches produced by the BC-Hoc* × Hag hybrids (i.e., 1/16: 6.25%), all of the resulting progeny shared the same maternal alleles for the 7 microsatellite loci examined (Table 2).This finding suggests that the haploid gametes were inherited hemiclonally in this clutch.In the remaining 15 clutches, either of the two maternal alleles were found alternatively in the resulting progeny, suggesting a recombinant mode of gamete production (Table 3).

Hybridogenesis expressed through genomic dissimilarity and genetic factors
Genomic heterogeneity (genetic affinity) between parental species plays an important role in the occurrence of hybridogenesis [17], but it is insufficient to induce this reproductive mode, which necessitates the conditional expression of certain genetic factors [23,25].The functionality of these unidentified genetic factors is hindered by the high genetic affinity observed between homologous chromosomes.This is substantiated by the observation that BC-Hoc*, comprising Hoc*/Hoc, produced recombined gametes through normal meiosis [19,23].That is to say, BC-Hoc* individuals can be characterized as carriers of hemiclonal genes.
In the present study, hybrids (BC-Hoc* × Hag) of the recombinant generation (BC-Hoc*) and Hag have shown a 1/16 (6.25%) chance of undergoing hybridogenesis.In eggs produced by BC-Hoc*, the genetic factor responsible for inducing hybridogenesis would be inherited by random recombination.If this factor was encoded by a single gene, then approximately half of BC-Hoc* × Hag should mature into hybridogens.The marked discrepancy between the observed result and the initial prediction, the result indicates that the genetic factor responsible for inducing hybridogenesis involves multiple genes, which are located at sites that are susceptible to recombination (i.e., either at distant positions on the same chromosome or on different chromosomes) (Fig 3).Gametogenesis by hybridogenesis requires several extraordinary steps, namely elimination of the paternal genome and duplication of the maternal genome [10,[26][27][28][29].Although the specific genes that are associated with hybridogenesis were not identified in this study, it is suggested that hemiclonal genes regulate several processes, including recognition and elimination of the paternal genome and selective preservation of the maternal genome.
In the hybrids Poeciliopsis monacha-occidentalis and P. monachal-lucida, the observed rates of hemiclonal hybrids resulting from artificial crosses between parental species were also low at 30% and 7%, respectively, suggesting that reestablishing hybridogenesis in these species is also difficult [30].Consequently, it is considered that the genetic factors that induce hybridogenesis are present in other cases of hybridogenesis, as well as in the genus Hexagrammos.The genus Bacillus (stick insect) hybrids, Pelophylax (Rana) esculenta (frog) and Misgurnus anguillicaudatus (loaches) have been reported to have various reproductive modes [4,31,32].In these lineages also, more complex mechanisms may be required to determine the reproductive mode.

Longevity and genetic diversity in hemiclonal organisms
The present study employed artificial crosses to demonstrate that a new hemiclone lineage can arise through recombinant generations (BC-Hoc*).Genetic diversity is unlikely to increase in the hemiclonal lineages and deleterious mutations are expected to accumulate if hybridogens are persistently backcrossed with males of the paternal species.This accumulation of deleterious mutations can be mitigated through the emergence of a new hemiclonal lineage through the BC-Hoc* generation, which produces recombined gametes.The offspring between P. monachalucida hemiclonal hybrids, which inherit only P. monacha genes, and P. viriosa, a sympatric sister species to P. monacha, produce recombinant gametes [33,34].In addition, it has been suggested that P. monacha-lucida hybrids may have obtained genetic variation by backcrossing with P. monacha [35].That is, backcrossing between Hoc*/Hag and Hoc males could also potentially play an important role in increasing the genetic diversity of the hemiclonal lineage.Indeed, the occurrence of BC-Hoc* by natural mating between Hoc*/Hag and Hoc males has been inferred using a specific cytogenetic marker, with Hoc*/Hag individuals observed to backcross with Hag and Hoc individuals with almost even probability [20].In hybrids that employ (hemi)clonal reproduction, changes in the species of the sperm donor are referred to as "host switching" [25,36], and the mate choice among such hybrids is referred to as "two-way backcrossing (crossing of hemiclonal hybrids with males of both parental species)" [20].This study showed that BC-Hoc* (carrier) individuals cannot be morphologically identified to Hoc individuals.These results suggest that BC-Hoc* fits successfully into the Hoc population and that the Hoc* genome is almost certainly present within the Hoc gene pool in the wild.Regeneration of new hemiclonal lineages is arisen from the Hoc gene pool when a hybrid possesses specific set of genetic factors that induce hybridogenesis.It is expected that distinct variations from the previous hemiclonal lineage will exist within the regenerated hemiclonal lineage that experience one or more recombinant generations.Given that the gene set is hypothesized to be comprised of several diverse components, regeneration is expected to occur only rarely.Therefore, when hybridogenesis reoccurs, the lineage will retain renewed genetic diversity.The BC-Hoc* × Hag hybrids cannot be identified as hemiclonal hybrids (Hoc*/Hag), indicating that the hybrids produced by the regeneration event have fitted into the wild hybrid population.This interpretation is supported by the previous reports.Hoc*/Hag hybrids possess polyphyletic haplotypes in mtDNA genealogical tree [25], indicating multiple origins.Estimates of genetic diversity based on mtDNA haplotypes showed that the regeneration events described in the present study occurred several times over the last 2.2-3.6 million years, i.e., after contact between populations of Hoc and Hag [22,25].
A similar diversity of mtDNA haplotypes has been observed in extant hemiclonal hybrids of P. monachal-lucida (e.g., [37]).In P. esculentus, in which hemiclonal males have also been reported, mtDNA exchange between parental species has been observed through backcrosses [38,39].Although classification of the genetic factors responsible for inducing hemiclones has yet to be elucidated in these taxa, the regeneration of new hemiclonal lineages through recombining generations, like BC-Hoc, could be one of the major keys to evolutionary success.

Fig 1 .
Fig 1. Lineages of the Hexagrammos hybrids referred to in the present study.If hybrids form between BC-Hoc and Hag, then hybridogenesis would be perpetuated by the inheritance of genetic factors that induce hybridogenesis; in the absence of such an inheritance pattern, recombinant reproduction would occur.Hoc and Hag represent H. octogrammus and H.agrammus, respectively.Blue and orange indicate the genomes of H. octogrammus and H. agrammus, respectively.https://doi.org/10.1371/journal.pone.0304772.g001

Fig 3 .
Fig 3. Inheritance pattern of recombination in three hemiclonal genes during gametogenesis.During gametogenesis of BC-Hoc*, chromosomes containing multiple hemiclonal genes (solid red circles) inherited from Hoc*/Hag recombine with homologous chromosomes inherited from Hoc.If all randomly rearranged hemiclonal genes are inherited, then new hemiclonal lineages are regenerated.Homologous chromosomes are comprised of either two solid or two stippled chromosomes.Hoc and Hag represent H. octogrammus and H.agrammus, respectively.Blue and orange indicate H. octogrammus and H. agrammus, respectively.https://doi.org/10.1371/journal.pone.0304772.g003

Table 3 .
(Continued)Green and Red-Orange indicated alleles inherited from the mother and father, respectively.Alleles present in both parents are shown in grey. https://doi.org/10.1371/journal.pone.0304772.t003